Association of the Polymorphisms of Vitamin D Receptor and Aggrecan Genes with Degenerative Disc Disease

Abstract

The aim of this study was to investigate the association between the polymorphisms of the vitamin D receptor (VDR) and aggrecan genes and degenerative disc disease in young Turkish patients. Aggrecan and VDR proteins are the main components of bone and cartilage. In our study, the polymorphisms of the VDR and aggrecan genes were investigated in a total of 300 individuals regarding disc degeneration and herniation. An association was found in the patients having VDR gene TT, Tt, FF, and Ff genotypes with the protrusion type of disc herniation, whereas the patients having tt and ff genotypes were associated with extrusion/sequestration types of the disease. Also, an association was observed between TT and FF genotypes of the VDR gene and mild forms of disc degeneration; and tt, ff, and Ff genotypes and severe forms of the disease. There was also an association between shorter, normal, and longer alleles of the aggrecan gene and a protrusion type of disc herniation. An association was found between short alleles and multilevel and severe disc degeneration, as well as normal and long alleles and mild disc degeneration. This study revealed that the polymorphisms of the VDR and aggrecan genes are associated with disc degeneration and herniation.

Introduction

Lumbar degenerative disc disease is one of the common causes of low back pain. Disc degeneration and disc herniation contribute to the development of low back pain and sciatica. Although the etiology of degenerative disc disease is complex, it has been suggested that some environmental factors, such as physical loading (Kujala et al., 1996), driving motor vehicles (Kelsey et al., 1984), vibration trauma (Pope and Hansson, 1992), and smoking (Kelsey et al., 1984), may play a role. On the other hand, it has been suggested that a genetic factor or familial predisposition contributes to the development of degenerative disc disease (Matsui et al., 1998). A number of genes have been associated with degenerative disc disease, including genes coding for collagen I, IX, and XI (Noponen-Hietala et al., 2003; Tilkeridis et al., 2005), aggrecan (Kawaguchi et al., 1999; Roughley et al., 2006; Solovieva et al., 2007), vitamin D receptor (VDR) (Jones et al., 1998; Videman et al., 1998, 2001; Kawaguchi et al., 2002; Cheung et al., 2006), matrix metalloproteinase-3 (Takahashi et al., 2001), and estrogen receptor alpha (Kawaguchi et al., 2002).

The polymorphisms of the VDR gene (FokI and TaqI) are thought to contribute to common disorders, such as osteoporosis (Riggs, 1997), osteoarthritis (Keen et al., 1997), and degenerative disc disease (Jones et al., 1998; Videman et al., 1998, 2001; Kawaguchi et al., 2002; Cheung et al., 2006). The VDR gene is located on chromosome 12q12-q14. VDR was the first reported gene associated with degenerative disc disease in a study of monozygotic twins in Finns with FokI and TaqI genotypes (Videman et al., 1998). This association was later confirmed in the studies of Japanese and southern Chinese populations (Kawaguchi et al., 2002; Cheung et al., 2006).

Intervertebral discs contain an abundant extracellular matrix of a fibrillar collagen network and proteoglycans. Proteoglycans are responsible for the high water content and play a critical role in the load carriage function of the disc. Aggrecan is the major proteoglycan component in cartilage and intervertebral disc. The aggrecan gene is located on chromosome 15q26. There have been very few studies of the association between the aggrecan gene variable numbers of tandem repeat (VNTR) polymorphism and degenerative disc disease. First, an association of the shorter alleles of aggrecan with an increased risk of severe and multilevel disc degeneration in young Japanese women was reported (Kawaguchi et al., 1999). Solovieva et al. (2007) found that there was a protective effect of the aggrecan alleles with 25 repeats and longer alleles on disc degeneration in Finnish men.

The aim of this study was to examine the association between the polymorphisms of the VDR and aggrecan genes and degenerative disc disease in a young Turkish population.

Materials and Methods

Participants

The participants were 300 adults aged 20-30 years. We attempted to exclude any environmental factor that might be related to lumbar disc disease. None of the participants was involved in physical loading, vibration trauma, ageing, and smoking. The subjects of this study included those with low back pain from the departments of neurosurgery in two different cities (Afyonkarahisar and Konya) of Turkey. All participants had a magnetic resonance imaging (MRI) scan. According to degenerated or nondegenerated intervertebral disc findings in lumbar MRI, the participants were divided into two groups each of 150 patients and 150 controls.

This study was approved by the Human Ethics Committee of Kocatepe University (Afyonkarahisar, Turkey). Informed consent was obtained from the participants before they were asked to join the study.

MRI analysis

Lumbar sagittal MRI was performed with a 5 mm slice thickness. A T2-weighted image with a repetition of 2500 ms and an echo time of 90 ms of the lumbar spine was taken in all the participants. The signal intensity of nucleus pulposus of the discs L2-L3, L3-L4, L4-L5, and L5-S1 was usually estimated independently by three observers. The grade of disc degeneration was determined according to Schneiderman's four-grade classification: Grade 1 (normal), normal signal intensity; Grade 2 (intermediate), heterogeneous decreased signal intensity; Grade 3 (marked), diffuse loss of signal; and Grade 4 (absent), signal void (Schneiderman et al., 1987). The types of disc herniation were evaluated by the criteria of MacNab's classification: normal disc, protrusion type, extrusion type, and sequestration type (Wiesel et al., 1996).

According to the data obtained, the levels of degeneration were categorized as Level 1, Level 2, and Level ≥3 (multilevel), whereas the grades of degeneration were classified as absent, Grades I and II (mild), and Grades III and IV (severe). In addition, the types of disc herniation were divided into following groups: absent, protrusion, and extrusion/sequestration type disc herniation.

Genomic DNA analysis

Peripheral blood was collected and genomic DNA was extracted from the samples. The polymorphisms of the VDR and aggrecan genes were detected using a polymerase chain reaction (PCR) assay. Detection of the TaqI and FokI restriction fragment length polymorphisms of the VDR gene was provided with use of forward and reverse primers in exon 2 (5′-AGCTGGCCCTGGCACTGACTCTGCTCT-3′ and 5′-ATGGAAACACCTTGCTTCTTCTCCCTC-3′) and another in exon 9 (5′-CAGAGCATGGACAGGGAGCAAG-3′ and 5′-CGGCAGCGGATGTACGTCTGCAG-3′). Amplification of the PCR was carried out in a 50 μL solution containing 10 pmol each of the sense and antisense primers, 5 μL genomic DNA, 25 mM diethylnitrophenyl thiophosphate, 0.5 μL of Taq DNA polymerase, 25 mM MgCl₂, and 10 × PCR buffer. Then the PCR products were digested with the endonucleases TaqI and FokI and separated on 2% agarose gels and visualized by ethidium bromide staining.

The sequences of sense and antisense primers for PCR of the aggrecan gene VNTR region analyzed were 5′-TAGAGGGCTCTGCCTCTGGAGTTG-3′ and 5′-AGGTCCCCTACCGCAGAGGTAGAA-3′, respectively. Amplification of PCR was carried out in a 50 mL solution containing 10 pmol each of the sense and antisense primers, 5 μL genomic DNA, 25 mM diethylnitrophenyl thiophosphate, 0.5 μL of Taq DNA polymerase, 25 mM MgCl₂, and 10 × PCR buffer. The PCR products were separated on 1.5% agarose gels and stained with ethidium bromide.

Statistical analysis

The chi-square test was performed to assess the trend of the aggrecan gene VNTR alleles and the restriction fragment length polymorphism genotypes of VDR gene regarding various parameters of disc degeneration: the number of the degenerated discs, the severity of the disc degeneration, and the type of the disc herniation. A p-value of less than 0.05 was considered statistically significant.

Results

The allele frequencies of VDR gene are shown in Table 1. We compared total T, t and F, f alleles between the patient and control groups, and the difference was not significant (p = 0.881, p = 0.443, respectively). A genotyping of the aggrecan gene VNTR polymorphism among 300 individuals resulted in identification of 11 alleles. The frequencies of these alleles in the studied population are presented in Table 2. There were no participants with alleles A18 and A20. A27 allele was the most common form, together with alleles A26 and A28.

Table 1.

Frequency of the Vitamin D Receptor Gene (TaqI and FokI) Alleles in Patients and Controls

Polymorphism	Alleles	Patients	Controls
TaqI	T	0.66	0.67
	t	0.34	0.33
FokI	F	0.72	0.67
	f	0.28	0.33

Table 2.

Frequency of the Aggrecan Gene Variable Numbers of Tandem Repeat Alleles in Patients and Controls

Alleles	Patients	Controls	Frequency (%)
A13 (775 bp)	4	0	0.7
A19 (1117 bp)	2	0	0.3
A21 (1231 bp)	13	2	2.5
A22 (1288 bp)	26	24	8.3
A25 (1459 bp)	28	29	9.5
A26 (1516 bp)	43	69	18.7
A27 (1573 bp)	90	74	27
A28 (1630 bp)	63	68	21.8
A29 (1687 bp)	30	33	10.5
A32 (1858 bp)	1	0	0.2
A33 (1915 bp)	0	1	0.2

The distribution of each genotype among the patients with disc degeneration and disc herniation was compared with that among the controls with normal discs (Table 3). A significant difference was found in the distribution of TaqI and FokI genotypes of the VDR gene with respect to the type of disc herniation. The TT, Tt, FF, and Ff genotypes were associated with protrusion type of disc herniation (p < 0.001). A significant association was found between tt and ff genotypes and extrusion and sequestration types of disc herniation (p = 0.023, p = 0.008, respectively). With respect to the number and grade of disc degeneration, a significant difference was found in the distribution of TaqI and FokI genotypes of the VDR gene. The TT and FF genotypes were associated with Grades I and II (mild) degeneration (p < 0.001, p < 0.001, respectively), whereas the tt, Ff, and ff genotypes were associated with Grades III and IV (severe) degeneration (p = 0.048, p = 0.004, p = 0.041, respectively).

Table 3.

Distribution of TaqI and FokI Genotypes of the Vitamin D Receptor Gene with Respect to the Grade and Level of Disc Degeneration and Type of Disc Herniation

	Level of disc degeneration			Grade of disc degeneration		Type of disc herniation
Genotype	1	2	≥3	Grades I and II (mild)	Grades III and IV (severe)	Absent	Protrusion	Extrusion/sequestration
TT	29	26	10	61	27	27	45	6
Tt	18	35	14	51	59	11	48	29
tt	4	6	8	10	22	4	9	16
FF	34	39	8	85	33	35	60	8
Ff	13	24	15	28	54	6	33	30
ff	4	4	9	9	20	1	9	13

The aggrecan alleles in the participants were divided into three groups: A13-26 (shorter alleles), A27 (normal lengthy alleles), and A28-33 (longer alleles) (Table 4) (Doege et al., 1997; Kawaguchi et al., 1999; Solovieva et al., 2007). The distribution was significantly different between protrusion type of disc herniation and A13-26, A27, and A28-33 allele groups (p < 0.001, p < 0.001, p = 0.001, respectively). The distributions of each allele group were compared between the participants with disc degeneration and those with normal discs. The distribution was significantly different among the two groups, with an overrepresentation of shorter alleles, which was 13-26 repeats in cases with severe disc degeneration (p = 0.044). A significant association was found between A13-26 and A27 alleles and multilevel disc degeneration (p = 0.018, p < 0.001, respectively).

Table 4.

Distribution of Aggrecan Gene Variable Numbers of Tandem Repeat Alleles with Respect to the Grade and Level of Disc Degeneration and Type of Disc Herniation

	Level of disc degeneration			Grade of disc degeneration		Type of disc herniation
Alleles	1	2	≥3	Grades I and II (mild)	Grades III and IV (severe)	Absent	Protrusion	Extrusion/sequestration
A13-26	32	53	30	76	103	27	82	45
A27	37	43	10	81	53	34	66	25
A28-33	33	38	24	87	59	23	56	32

The combinations of genotypes were defined by the genotypes of the VDR and aggrecan genes. The distributions of each combined genotype were compared with the type of the disc herniation, the level of the degenerated discs, and the grade of the disc degeneration. However, no significant difference was seen in the effect of VDR and aggrecan gene polymorphisms on phenotypes either individually or in combination regarding the type of the disc herniation, the level of the degenerated discs, and the grade of the disc degeneration.

Discussion

Lumbar degenerative disc disease is one of the most frequently seen spine diseases. Age, occupation with heavy physical load, motor vehicle driving, vibration, and smoking have been reported as risk factors for structural damage of intervertebral disc (Kelsey et al., 1984; Pope and Hansson, 1992; Kujala et al., 1996). However, recent studies have shown an association between genetic influences and disc degeneration. Further, for some genetic influences and some environmental factors, gene-gene interactions and gene-environment interactions may exist. To our knowledge, this is the first genetic study that investigated the association between VDR and aggrecan genes polymorphisms with degenerative disc disease in the Turkish population.

Previous findings of the association of VDR and aggrecan genes polymorphisms with degenerative disc disease have been replicated in an ethnically distinct population, the Turkish population.

VDR is steroid nuclear receptor, better known to have a role in normal bone mineralization and remodeling. VDR expression was reported in chondrocytes and is thought to be involved in differentiation, proliferation, and maturation of cartilage (Balmain et al., 1993). In addition, vitamin D has influenced proteoglycan synthesis (Corvol et al., 1981). Beck and Silve (2001) demonstrated that vitamin D status modulates plasma sulfate concentrations, and as a result, it may influence the amount of inorganic sulfate available for intracellular sulfation of proteoglycans.

Different investigations have suggested that allele frequencies of VDR gene are different among ethnic groups. In our study and Finns (Videman et al., 1998) the t allele was present in 33% of the population versus 13% in Japanese (Kawaguchi et al., 2002) and 3.71% in the southern Chinese (Cheung et al., 2006). On the other hand, in our study, f allele frequency was 30% versus 40% in Finns population.

Kawaguchi et al. (2002) reported the association between TaqI genotypes and disc herniation. These findings were similar to our results. By contrast, no association was found between TaqI and FokI genotypes and disc herniation by Videman et al. (1998). On the other hand, Videman et al. found that quantitatively assessed signal intensities in thoracic and lumbar discs in men with the tt genotype and in men with the Tt genotype were worse (12.9% and 4.5%, respectively) than those in men with the TT genotype. In this study, a similar pattern was also found between disc signal intensity and FokI genotypes; men with the ff and Ff had mean signal intensities that were 9.3% and 4.3% lower, respectively, than those in men with FF genotype. Kawaguchi et al. reported that subjects with the Tt genotype were more frequently found to have multilevel and severe disc degeneration than the subjects with the TT genotype. In a southern Chinese study, Cheung et al. (2006) showed an association between the Tt and tt genotypes and degenerative disc disease. Our findings were consistent with these results.

Aggrecan, a large aggregating proteoglycan, is the major structural component of intervertebral disc. Aggrecan provides intervertebral disc with the ability to resist compressive loads and osmotic pressure. It has a long core protein, the glycosaminoglycan attachment region, to which numerous keratan sulfate and chondroitin sulfate chains are attached. Exon 12 of aggrecan gene codes for entire glycosaminoglycan-binding region. A VNTR polymorphism in this region of the aggrecan gene may decrease a number of chondroitin sulfate chains and may predispose to intervertebral disc degeneration.

A total of 13 different alleles of VNTR polymorphism have been observed, with repeat numbers ranging from 13 to 33 (Doege et al., 1997; Kirk et al., 2003). We did not find alleles that contained 18 and 20 repeats, whereas alleles A27, A28, and A26 (containing 27, 28, and 26 repeats, respectively) were overrepresented in the individuals. The frequency of the alleles found in other studies was different between populations. In the Finnish population, different frequencies of the alleles were found in two different studies (six and nine different alleles, respectively) (Videman et al., 1998, 2001). In young Japanese versus Canadian populations, 8 and 13 repetitive alleles were reported, respectively (Kawaguchi et al., 1999; Roughley et al., 2006).

There have been very few studies of the aggrecan gene VNTR polymorphism. Kawaguchi et al. (1999) previously reported that subjects with polymorphism of the aggrecan gene are at risk for early disc degeneration. Multilevel and severe disc degeneration was present more often in young Japanese women with shorter alleles than in those with longer alleles. Our results were similar to that in analyses in Japanese population. On the other hand, no difference in the incidence of disc degeneration in individuals with at least one allele with less than 25 repeats was found by Roughley et al. (2006). Solovieva et al. (2007) indicated that there was a protective effect of the alleles with 25 repeats and longer alleles (with 27 or more repeats) on disc degeneration. The other study's findings indicated that allele A27 provides protection from hand osteoarthritis but alleles shorter or longer than this may predispose subjects to the disease (Kämäräinen et al., 2006). On the other hand, no association between the VNTR polymorphism and degenerative lumbar spinal stenosis was found by Noponen-Hietala et al. (2003).

In a Japanese population, no significant association was found between the alleles and the number or type of disc herniation by Kawaguchi et al. (1999). By contrast, our study revealed that protrusion types of disc herniation were present in the participants with shorter, normal, and longer VNTR alleles of aggrecan gene.

Our findings suggested that the VDR and aggrecan gene polymorphisms might be a risk for disc degeneration and disc herniation at an early age. We first reported the association between the VDR and aggrecan gene polymorphisms and degenerative disc disease in Turkish population. A limitation of our study was the relatively small sample size. This study needs to be replicated in larger population samples and the changes in mRNA and protein levels of VDR and aggrecan of the intervertebral discs with the grade of disc degeneration have to be investigated.

Footnotes

Acknowledgment

This work was supported by the research fund of the Afyon Kocatepe University (project 06.TIP.27).

Disclosure Statement

No competing financial interests exist.

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